The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1δ/ε

Yasunori Sugiyama, Naoya Hatano, Noriyuki Sueyoshi, Isao Suetake, Shoji Tajima, Eiji Kinoshita, Emiko Kinoshita-Kikuta, Tohru Koike, Isamu Kameshita

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Dnmt1 (DNA methyltansferase 1) is an enzyme that recognizes and methylates hemimethylated DNA during DNA replication to maintain methylation patterns. The N-terminal region of Dnmt1 is known to form an independent domain structure that interacts with various regulatory proteins and DNA. In the present study, we investigated protein kinases in the mouse brain that could bind and phosphorylate the N-terminal regulatory domain of Dnmt1. A protein fraction containing protein kinase activity for phosphorylation of Dnmt1(1-290) was prepared using Dnmt1(1-290)-affinity, DNA-cellulose and gel-filtration columns. When the proteins in this fraction were analysed by LC-MS/MS (liquid chromatography tandem MS), CK1δ/ε (casein kinase 1δ/ε) was the only protein kinase identified. Recombinant CK1δ/ε was found to bind to the N-terminal domain of Dnmt1 and significantly phosphorylated this domain, especially in the presence of DNA. Phosphorylation analyses using various truncation and point mutants of Dnmt1 revealed that the major priming site phosphorylated by CK1δ/ε was Ser146, and that subsequent phosphorylation at other sites may occur after phosphorylation of the priming site. When the DNA-binding activity of phosphorylated Dnmt1 was compared with that of the non-phosphorylated form, phosphorylation of Dnmt1 was found to decrease the affinity for DNA. These results suggest that CK1δ/ε binds to and phosphorylates the N-terminal domain of Dnmt1 and regulates Dnmt1 function by reducing the DNA-binding activity.

Original languageEnglish
Pages (from-to)489-497
Number of pages9
JournalBiochemical Journal
Volume427
Issue number3
DOIs
Publication statusPublished - May 1 2010

Keywords

  • Casein kinase 1 (CK1)
  • DNA methylation
  • DNA methyltransferase
  • DNA-binding activity
  • Domain structure
  • Protein phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Sugiyama, Y., Hatano, N., Sueyoshi, N., Suetake, I., Tajima, S., Kinoshita, E., Kinoshita-Kikuta, E., Koike, T., & Kameshita, I. (2010). The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1δ/ε. Biochemical Journal, 427(3), 489-497. https://doi.org/10.1042/BJ20091856